METHOD FOR TREATING MEDIATED VASCULAR DISORDERS BY THE VASCULAR ENDOTHELIAL GROWTH FACTOR
This invention relates to the use of 2-amino-3-benzoylbenzenacetic acid (amfenac) to treat or prevent vascular disorders mediated by vascular endothelial growth factor (VEGF).
Background of the Invention It has previously been shown that certain non-steroidal anti-inflammatory drugs (NSAIDs) can inhibit the formation of new blood vessels (angiogenesis) under pathological conditions, as well as vascular filtration in certain models of inflammation. The ability of most NSAIDs to affect vascular permeability and angiogenesis seem to be associated with their ability to block the cyclo-oxygenase enzymes (COX-1 and 2). Blockade of COX-1 and 2 is associated with a decrease in inflammatory mediators, such as PGE2. In addition, it appears that the inhibition of PGE2 results in decreased expression and production of vascular endothelial growth factor (VEGF). VEGF is known to produce vascular filtration and angiogenesis in the eye of pre-clinical models. Also, increased levels of VEGF have been found in neovascular tissues and extracellular fluid from the eyes of patients with diabetic retinopathy and age-related macular degeneration. In this way, NSAIDs can inhibit vascular filtration and angiogenesis by modulating PGE2 levels and their effects on VEGF expression and activity. This theory is supported by work involving animal tumor models that demonstrate that systemic administration of COX-2 inhibitors decreases PGE2 and tissue levels of VEGF thereby avoiding tumor-induced angiogenesis. In these models, the activity of VEGF and angiogenesis is restored by adding exogenous PGE2 during continuous COX-2 blockade. However, NSAIDs appear to have variable activity in animal models of ocular neovascularization (NV), in which selective COX inhibitors do not appear to inhibit choroidal neovascularization. In fact, these studies have called into question the role of COX-1 and / or COX-2 in the development of CNV. 3-Benzoylphenylacetic acid and certain of its derivatives are known to possess anti-inflammatory activity. U.S. Patent Nos. 4,254,146, 4,045,576, 4,126,635, and 4,503,073, and U.S. Patent Applications Nos. 2,071,086A, 2,093,027A describe various 3-benzoylphenylacetic acids, salts and esters, and hydrates thereof, which have anti-activity. -inflammatory U.S. Patent No. 4,568,695 discloses 2-amino-3-benzoylphenylethyl alcohols having anti-inflammatory activity. U.S. Patent No. 4,313,949 describes 2-amino-3-benzoylphenylacetamides having anti-inflammatory activity. Certain derivatives of 2-amino-3-benzoylbenzeneacetic acid (amfenac) and 2-amino-3- (4-chloro-benzoyl) benzeneacetic acid have also been evaluated by Walsh et al., J. Med Chem., 33: 2296- 2304 (1990), in an attempt to discover non-steroidal anti-inflammatory prodrugs with minimal or no gastrointestinal side effects in oral administration. U.S. Patent No. 4,683,242 teaches the transdermal administration of 2-amino-3-benzoylphenylacetic acids, salts and esters, and hydrates and alcoholates thereof to control inflammation and relieve pain. U.S. Patent No. 4,910,225 teaches certain benzoylphenylacetic acids for local administration to control ophthalmic, nasal or otic inflammation of the ear. Only acetic acids are described in the '225 patent; Esters or amides are not mentioned or taught as anti-inflammatory agents for local administration to eyes, nose and ears. U.S. Patent No. 5,475,034 discloses topically administrable compositions containing certain amide and ester derivatives of 3-benzoylphenylacetic acid, including nepafenac, useful for treating ophthalmic inflammatory disorders and ocular pain. According to the patent 034 in column 15, lines 35-39, "such or such disorders include, but are not limited to, sclerosis by uveitis, episcleritis, keratitis, surgically induced inflammation and endofthalmitis". US Patent No. 6,066,671 describes the topical use of certain 3-benzoylphenylacetic acid amide and ester derivatives, including nepafenac, to treat GLC1A glaucoma. In the commonly owned North American application Serial No. 09 / 929,381, it was found that certain 3-benzoylphenylacetic acids and derivatives are useful for treating disorders related to angiogenesis.
Detailed Description of the Invention Posterior segment neovascularization (NV) is the vision-threatening pathology responsible for the two most common causes of blindness in developed countries: macular degeneration related to exudative age (AMD) and proliferative diabetic retinopathy. Currently the only approved treatments for posterior segment NV that occurs in exudative ADM is laser photocoagulation or photodynamic therapy with Visudyne; both therapies involve occlusion of affected vasculature that results in laser-induced damage located in the retina. Surgical interventions with vitrectomy and membrane removal are the only options currently available for patients with prolif- rative diabetic retinopathy. No strictly pharmacological treatment has been approved for use against posterior segment NV. In addition to changes in retinal microvasculature induced by hyperglycemia in diabetic patients that lead to macular edema, neovascular membrane proliferation is also associated with vascular filtration and retinal edema. Where the edema involves the macula, worsening visual acuity. In diabetic retinopathy, macular edema is the major cause of vision loss. Since laser photocoagulation is used for angiogenic disorders to stabilize or resolve the edematous condition. Unfortunately, laser photocoagulation is a cytodestructive procedure, which, while preventing further edema from developing, will alter the visual field of the affected eye. An effective pharmacological therapy for posterior segment NV and edema will probably provide efficacy to the patient, thus avoiding invasive surgical procedures or laser damage. The effective treatment of NV would improve the patient's quality of life and productivity within society. Also, the social costs associated with providing assistance and health care to the blind could be dramatically reduced. Amfenac is an NSAID that is known to potentially inhibit the activity of COX-1 and COX-2 enzymes. Unexpectedly, amfenac was found to inhibit VEGF-induced cell proliferation and capillarity tube formation in a dose-response fashion using a bovine retinal microvascular endothelial cell assay. To our knowledge, this blockade in VEGF effects by NSAIDs occurs independently of COX inhibition, ie, the ability to block the proangiogenic signal normally produced by VEGF, is unique with respect to amfenac against other NSAIDs. This unique activity may help to explain, in part, the previous findings that the topical nepafenac (the amfenac prodrug) inhibits choroidal NV in a mouse model, where VOLTAREN® topical and ACULAR® had no effect. If this novel anti-angiogenic activity occurs in men, amfenac (and topical nepafenac) could be used to more effectively treat diseases that involve VEGF signaling and in disease states where other NSAIDs could probably be less effective. Ophthalmic disorders associated with up-regulation of VEGF that are potential indications for amfenac (topical nepafenac) would include macular degeneration related to exudative age, proliferative diabetic retinopathy, retinal vein occlusion, proliferative vitreoretinopathy, neovascular glaucoma, corneal angiogenesis, retinal microvasculopathy and retinal edema (macular). Again, because amfenac is the active metabolite of nepafenac, which has the ability to reach the posterior segment after topical corneal application in pre-clinical models, it is possible to treat these eye disorders mediated by VEGF using topical ocular administration of nepafenac . According to the present invention, a therapeutically effective amount of a nepafenac is administered topically to an eye while local or systemic administration of amfenac will be used to treat and / or prevent vascular disorders mediated by VEGF. The doses of amfenac or nepafenac used in the treatment or prevention of vascular abnormalities medicated with VEGF will depend on the type of abnormality that is to be avoided or treated., the age and body weight of the patient, and the form of the preparation / route of administration. The compositions intended for ophthalmic administration will normally contain nepafenac in an amount from about 0.001 to about 4.0% (w / v), preferably from about 0.01 to about 0.5% (w / v), with 1-2 drops once to several times at day. Also, the representative doses for other forms of preparations are approximately 1-100 mg of amfenac / day / adult for injections or local administration and approximately 10-1000 mg of amfenac / adult for oral preparations, each administered once to several times up to date. Additional therapeutic agents can be added to supplement the use of nepafenac or amfenac. The following examples are presented to illustrate various aspects of the present invention, but are not intended to limit the scope of the invention in any respect. The percentages are expressed on a weight / volume basis.
Example 1: The following formulations are representative of the topical compositions useful in the present invention.
Formulation 1 Nepafenac 0.01-0.5% Polysorbate 80 0.01% Benzalkonium Chloride 0.01% + 10% excess Disodium EDTA 0.1% Sodium Phosphate Monobasic 0.03% Sodium Phosphate Dibasic 0.1% Sodium Chloride c.s. 290-300 mOsm / Kg Adjust pH with NaOH and / or HCl pH 4.2-7.4 Water q.s. 100% Formulation 2 Nepafenac 0.01-0.5% Hydroxypropylmethylcellulose 0.5% Polysorbate 80 0.01% Benzalkonium Chloride 0.01% + 5% excess EDTA disodium 0.01% Sodium Phosphate Dibasic 0.2% Sodium Chloride is. 290-300 mOsm / Kg Adjust pH with NaOH and / or HCl pH 4.2-7.4 Water q.s. 100%
Formulation 3 Nepafenac 0.1 + 6% excess Carbopol 97 P 0.08% Tyloxapol 0.01% Glycerin 2.4% Disodium EDTA 0.01% Benzalkonium Chloride 0.01% pH adjustment with NaOH and / or HCl pH 7.5 ± 0.2 Water C.S. 100%
Example 2 Effect of AL06295A (Amfenac) on BRMEC Proliferation (Bovine Microvascular Endothelial Bovine Cell) The VEGF-induced proliferation of BRMEC was measured using a modified MTT assay, plated at 3 x 103 BRMEC in a fibronectin matrix / hyaluronic acid in 96-well plates (Corning). The growth medium was added for two days, followed by serum-free medium (SFM) overnight, then by means of a test containing 0 or 25 ng / ml of VEGF, in 100 μ? of SMF. After 24 hours at 37 ° C / 5% C02, 25 μ? of MTT of (3- (, 5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) was added to each well and incubated for 4 hours. Then 100 μ? of lysis buffer (20% SDS in 50:50 DF: H20 + 20% acetic acid and 0.05% HC1) to each well, and the plates were incubated overnight at 37 ° C and read (Molecular Devices SPECTRAmax 190; Sunnyvale, CA) at 570 nm. For experiments using AL06295, 25 ng / ml of VEGF was combined with the compound at 0.1, 0.3, 1.0 or 3 μ ?. The results show that 1 and 3 μ? of amfenac dose significantly reduce proliferation of VRMEC induced by VEGF, see Figure 1.
Example 3 Effect of AL06295A (Amfenac) on BRMEC Tube Formation An 8 vol mixture of Vitrogen 100 (Cohesion, Palo Alto, CA), 1 vol. 0.2 N NaOH, and 1 vol. of 10 x of an RPMI-1640 medium containing 5 μg / ml of fibronectin and 5 μg / ml of laminin was prepared and 400 μ? was added to each well of a 24-well plate. After incubation for 3 hours at 37 ° C to solidify the gel, 104 BRMEC were added to each well and incubated in growth medium for 3 days. Then the medium was carefully aspirated and 200 μ? of the gel solution on top of the cells and incubated at 37 ° C for 1 hour. After the addition of the growth medium for 24 hours, 2 ml of test medium containing a serum-free medium (SF) plus VEGF or SF medium plus VEGF and AL06295A was added to each well. The gels were evaluated 24 hours later. For quantitative analysis, six fields per treatment group were chosen from the areas containing tubes; Seven wells were used for each treatment. The lengths of the tubes were measured in digitized images, and the data are expressed in Figure 2 as the total length per field of view in μp ?. The results show that all amfenac doses significantly and potentially inhibit VEGF-induced capillary tube formation in the BRMEC. This invention has been described for reference to certain preferred embodiments; however, it must be understood that it may be involved in other specific forms or variations thereof without departing from its special or essential characteristics. The embodiments described above are therefore considered to be illustrative in all respects and not restrictive, within the scope of the invention which is indicated by the appended claims rather than by the foregoing description.